Development of an efficient and reproducible in vitro regeneration and transformation protocol for tropical maize (Zea mays L.) using mature seed‑derived nodal explants.

Abstract

Maize is an important crop for billions of people globally. The existing immature embryo-based regeneration protocol of maize has major limitations due to the non-availability of explants throughout the year, limited durability for culturing, and its laborious nature. Mature embryos, especially in tropical maize, are considered recalcitrant towards tissue culture. Therefore, standardization of a robust regeneration and transformation protocol in tropical maize using mature embryos or seeds as starting material is long envisaged. Considering this, in this study, 28 diverse tropical maize genotypes were evaluated for their embryogenic callus induction potential using two different explants (nodal explants and split embryo region) under two different callusing media. Out of 28 genotypes, better callus induction was achieved in four genotypes (BML 6, DHM 117, DMRH 1301, and DMRH 1308) from nodal explants. Further, in vitro regeneration was standardized using 22 different combinations of various auxins and cytokinins. Out of 28 genotypes, two recently commercialized and high-yielding cultivars (DMRH 1301 and DMRH 1308) demonstrated the best callusing and regeneration capability with an average regeneration percentage of 60.4% and 53.6%, respectively. Using the nodal explants-derived embryogenic calli, the genetic transformation was successfully carried out using the ‘Biolistic’ approach, and up to ~ 5% transformation efficiency was achieved. This efficient regeneration and transformation protocol can overcome the major limitations associated with the existing immature embryo-based protocol in tropical maize as mature seeds can be obtained easily in ample quantity round the year. Such a generalized and reproducible protocol has the potential to be a major tool for maize improvement using transgenic and genome-edited techniques.